1. Field of the Invention: This invention relates to the blood coagulation components Factors VII and VIIa, and to a process for producing a blood coagulation promoting product containing Factors VII and VIIa in high yield and relative purity and free from other coagulation factors.
2. Cross-reference: The present application is related to U.S. Pat. Nos. 4,470,969 and 4,473,553, both of which are owned with the present application by a common assignee. These related applications concern processes for producing coagulation factors VII and VIIa by contacting aqueous preparations containing the factors VII and VIIa with a lipoprotein binding adsorbent, contacting the resulting lipoprotein-poor solution with an adsorbent having selective affinity for calcium-binding coagulation factors and selectively eluting factors VII and VIIa therefrom. As the lipoprotein binding adsorbent, there is used in U.S. Pat. No. 4,470,969 a polyanionic adsorbent in combination with divalent metal ions whereas there is used in U.S. Pat. No. 4,473,553 polyethylene glycol.
3. Description of the Prior Art and Related Art: U.S. Pat. Nos. 3,717,708, 4,404,132 and 4,361,510 disclose a blood coagulation promoting complex concentrate product free of thrombin, heparin, activated Factor X, depressor activity, and anticomplement activity, and containing coagulation Factors II, VII, IX and X in non-activated form. More particularly, U.S. Pat. No. 3,717,708 discloses the product described above produced by a process comprising the steps of applying Cohn Supernatant I (also referred to as "Cohn Effluent I"), prepared by Method 6 from unmodified citrated human plasma, which contains Factors II, VII, IX and X, onto an anion exchange resin consisting of cross-linked dextran chains with diethyl-aminoethyl groups attached by ether linkages to the glucose units of the polysaccharide chains (e.g. DEAE Sephadex.RTM., Pharmacia Fine Chemicals, Inc.) and adsorbing thereon the given coagulation Factors; selectively eluting the anion exchange resin from the preceding step with ammonium bicarbonate solution of pH in the range of 7.3 to 8.2 and of increasing molarity between 0.3M and 0.75M; separating the eluate containing the given coagulation Factors; freezing the separated eluate and removing the amonium bicarbonate therefrom by lyophilizing the frozen fraction. U.S. Pat. Nos. 4,404,132 and 4,361,510, each arising out of a common parent application, now-abandoned, disclose improvements in the above-described process and product of U.S. Pat. No. 3,717,708 wherein the elution step is carried out with an aqueous solution containing a non-volatile salt and citrate ions in place of an aqueous solution of the volatile salt, ammonium bicarbonate, and wherein a combined ultrafiltration-diafiltration step is used in place of lyophilization to remove ammonium bicarbonate, these improvements providing a product having greater Factor IX specific activity than that of the product from U.S. Pat. No. 3,717,708.
Thomas, U.S. Pat. No. 4,287,180 discloses a method for treating a patient having an inhibitor of blood clotting factors by administering to the patient a therapeutically effective dose of an aqueous composition including, in units/ml, factor VIII correctional activity, about 2-35; prothrombin, about 1-10; thrombin, less than 0.003; factor VII, about 37-190; factor VIIa, about 8-80; factor IX, about 15-112; factor IX precursor, 0-30; factor X, about 1-30; and factor Xa, about 1-10.
Thomas, U.S. Pat. No. 4,357,321, a division of U.S. Pat. No. 4,287,180 mentioned above, discloses a method for treating a patient having an inhibitor of blood clotting factor by administering to the patient a therapeutically effective does of an aqueous composition of 37-110 units/ml of factor VII and 8-80 units/ml of factor VIIa.
In both of the foregoing Thomas patents, there is disclosed the use of Cohn plasma fractions I+II+III, I and III, II and III, III, III-0, IV-1, or IV-1 and IV-4. IV-1 is the preferred fraction and is disclosed in the working examples. The starting compositions are dissolved in a buffer or saline to a concentration of about 10% weight/volume at about 20.degree. C., screened for clotting factor activity to determine the degree of preexisting spontaneous activation, partially purified by adsorption onto a suitable, known prothrombin complex adsorbent (e.g. tribasic calcium phosphate or a diethylaminoethyl group substituted resin), followed by elution from the adsorbent in a volume of about 4% of the volume of the dissolved Cohn fraction, none of the volumes or temperatures being critical. Example 1 of the patent illustrates a typical manufacturing run for the preparation of an activated PCC (prothrombin complex) according to the invention of the patent wherein the starting material, a Cohn fraction IV-1 paste, is suspended in saline at a pH of 7.2; a resultant precipitate is allowed to settle; and a clear supernatant is obtained by centrifugation. Then calcium phosphate is added to the supernatant, the calcium phosphate adsorbed coagulation factors are recovered, the factors separated from calcium phosphate by vigorous mixing with a volume of 0.1M sodium citrate equal to 4% of the dissolved paste, and the resulting suspension is centrifuged and the supernatant is recovered. Following this, the coagulation factors in the supernatant are activated by contacting the supernatant with silica and the activated PCC is further purified by PEG precipitation steps followed by dissolution of the precipitate in 0.02M sodium citrate, sterile filtration and lyophilization.
Thomas, U.S. Pat. No. 4,382,083 and PCT publication WO No. 83/00016 which corresponds thereto, disclose a method for treating a patient having a clotting factor defect by administering a composition of an effective hemostatic amount of factor VIIa, which composition is uncontaminated by other activated blood clotting factors having sufficient activity alone to produce a hemostatic effect. Unactivated precursor forms of factors may be present in the compositions and the amounts of unactivated factors II, VII, IX and X typically range from about 1-10, 20-250, 0-30, and 1-30 units/ml, respectively. Preferably, the compositions are essentially free of factors IX and II but contain factors VII and/or X in the above concentrations. Suitable factor VIIa compositions for use according to the patented method can be produced according to the methods of Kisiel et al, Biochemistry, 16 (9), 4189 (1977) or Broze and Majerus, J. Biol. Chem., 255, 1242 (1980).
Kisiel et al, supra, disclose a 5-step procedure to isolate and purify factor VII from bovine plasma which involves barium sulfate adsorption and elution, DEAE Sephadex.RTM. batchwise adsorption and elution, benzamidine-agarose column chromatography, heparin-agarose column chromatography, and preparative polyacrylamide gel disc electrophoresis.
Broze and Majerus, supra, disclose the purification of human factor VII from human, citrated, fresh frozen plasma with platelets removed by barium citrate adsorption and elution and ammonium sulfate fractionation. The resulting solution was then treated to two successive QAE-Sephadex chromatographic purification steps followed by gel filtration on Sephadex G-100.
Bajaj et al, J. Biol. Chem., 356 (1), 253 (1981), disclose the purification of human factor VII from blood plasma using barium citrate adsorption, ammonium sulfate fractionation, DEAE-Sephadex chromatography, preparative polyacrylamide gel electrophoresis (PAGE), and SDS-PAGE.
Gladhaug et al, Biochim. Biophys. Acta, 215, 105 (1970), disclose the purification of factors VII and X from human serum using barium sulfate-Sephadex, DEAE-Sephadex, polyacrylamide gel filtration, and polyacrylamide gel disc electrophoresis.
Current thinking in the field to which this invention pertains is that there are two separate systems--intrinsic and extrinsic--which can promote clotting and can thereby participate in normal hemostasis. In factor VIII deficient persons, who also have circulating antibodies to the molecule, replacement therapy with factor VIII concentrates has been shown to be of limited or of no value, presumably because the intrinsic system in such persons has been largely inhibited. Treatment with prothrombin complex products, however, has had varying degrees of success in these patients; but the active principal in these materials has not, as yet been elucidated. It is not unlikely that these products function by simply enhancing the contribution of the extrinsic system to clotting, taking into consideration that they contain concentrated amounts of some of the extrinsic system enzymes, namely, factors II, VII and X, more specifically, these products may function as follows: Factor VII/VIIa levels are raised to the extent that appreciable levels of tissue factor VII/VIIa complex are formed. This complex can interact with both of factors IX and X and, if this occurs, a series of activations can arise. Activated factor X is known to be able to activate factor VII and factor IX reciprocally, to be autoactivatable, and to activate factor II. The net effect of any of these reactions would thus be a great increase of factor Xa and, hence, factor IIa activity, which would further result in subsequent clot formation.
Crucial to the above-described scheme is the concept that the activation remains localized to prevent systemic, uncontrolled clot formation. Under normal conditions, this danger is avoided probably because tissue factor may be fairly selectively exposed at the site of injury keeping the activations within a local environment and also because of the presence of circulating, naturally occurring protease inhibitors.
That activation can occur at all in the presence of these circulating inhibitors, even in a local environment, may be due to the reciprocal activations of factors VII, IX and X. Although it is generally accepted that the extrinsic system can function as described at least in vitro, it is not understood why the extrinsic system does not control bleeding in factor VIII deficient persons. It is conceivable that the extrinsic system under normal circumstances acts only as an auxiliary to the intrinsic system to promote clotting. While it may be that the levels of extrinsic system participants are high enough to repair certain kinds, or limited amounts of, physiologic damage, they may not be sufficient to correct the major bleeding episodes that occur in factor VIII or IX deficiencies. According to the above, factor VII would be a requirement in order for the described reciprocal activations to occur.
There is a need, then, for a process to produce factor VII and factor VIIa in relatively pure form, free of the other factors, II, IX and X, with which factors VII and VIIa are generally associated and isolated, for both experimental and therapeutic purposes.